**3. Results**

### **3.1. Patients' characteristics**

A total of 612 patients were evaluated, of which 123 fulfilled the eligibility criteria; finally, 100 had complete data to enter the analysis, 76 males and 24 females, median age 68 years (range 45-81). Diabetes mellitus was recorded in 26 of the 100 patients and hypertension in 66. Twenty patients had metabolic syndrome according to the national cholesterol education programmeadult treatment panel III (NCEP-ATP III) criteria [29]. Active smoking (defined as current of discontinued as far back as 5 years) was reported by 58 patients. Mean atorvastatin dose at baseline was 24.31±11.49 mg for group A and 20.62±10.39 mg for group B (p=0.1). By the end of the study period, the respective mean values were significantly increased to 30.45±16.27 mg for group A (p=0.044) and 28.75±17.57 mg for group B (p=0.007).

A significant correlation between LDL and oxLDL levels was detected (Pearson's correlation coefficient r=0.7, p<0.01) (Figure 6). Similar correlation was found between oxLDL and apoB

Oxidised Low Density Lipoprotein (LDL) Modification with Statin Therapy is Associated with...

http://dx.doi.org/10.5772/57188

133

Between smokers mean oxidized LDL was reduced from 60.68±24.09 mU/l at baseline to 45.84±24.89 mU/l at the end of study period (difference 14.84 mU/l, p = 0.0036). Similarly, between non-smokers it was reduced from 69.33±25.11 to 40.36±5.6 (difference 28.97, p<0.001). Non-smokers had approximately double decline of oxidized LDL levels compared to smokers. Carotid artery stenosis was reduced between smokers from 29.68±25.59% at baseline to 23.06±21.71% at 12 months (p = 0.002). Non-smokers also presented significant reduction of stenosis during the study period 24.67±26.22% vs 20±21.45%, p = 0.004). Non-smokers and smokers had similar decline of carotid stenosis in 12 months (6.61% vs 4.67%, Table 3).

> 0 1 2 3 4 Time periods of observation

TC: total cholesterol, LDL: low density lipoprotein cholesterol, oxLDL: oxidised LDL, TG: triglycerides, 0=baseline, 1=one

**Figure 5.** Time curve of change of total cholesterol, LDL cholesterol, triglycerides and oxidised LDL levels during the

In further analysis, the group of smokers was subdivided to mild (≤5 cigarettes/day), moderate (5 – 15 cigarettes/day) and heavy (≥15 cigarettes/day) smokers. The statistical significant reduction of oxidized LDL levels and degree of carotid stenosis was apparent in the subgroup of mild smokers (oxidized LDL at baseline 48.24±8.74 mU/l vs 41.54±9 mU/l at 12 months, p = 0.027 and stenosis at baseline 27.63±25.68% vs 23.42±21.74% at 12 months, p = 0.009), while it was not apparent in the subgroups of moderate and heavy smokers (oxidized LDL at baseline 86.82±37.7 mU/l vs 42.92±10.77 mU/l at 12 months, p = 0.077 and stenosis at baseline 34±31.9% vs 22±24.9% at 12 months, p = 0.186, for moderate smokers; respective values for oxidized LDL were 66.29±15.88 mU/l vs 34.81±5.48 mU/l, p = 0.06 and for stenosis 32.14±24.13% vs

Lipids alteration during observation period

TC LDL oxLDL TG

levels (r=0.65, p<0.001), while no significant correlation was shown with Lp-a.

50

observation period.

month, 2=three months, 3=six months, 4=twelve months.

100

150

mgr/dl

200

250

Each of the study group (A and B) comprised 50 patients. The two groups were comparable with regard of their baseline characteristics (table 1).

#### **3.2. Lipid profile and oxidised LDL**

Mean serum TC, LDL-cholesterol, TG, Lp-a, homocysteine, HDL-cholesterol, and oxLDL were significantly reduced at 12 months compared to baseline (table 2). Specifically, mean oxLDL dropped from 62.26±22.03 mU/l to 44.49±21.75 (p<0.001). A marked decrease was noticed during the first 6 months and a plateau thereafter (Figure 5).


Pts: patients, DM: diabetes mellitus, HTN: arterial hypertension, MS: metabolic syndrome, SD: standard deviation, LDL: low density lipoprotein, HDL: high density lipoprotein, CAD: coronary artery disease

**Table 1.** Study population baseline characteristics

A significant correlation between LDL and oxLDL levels was detected (Pearson's correlation coefficient r=0.7, p<0.01) (Figure 6). Similar correlation was found between oxLDL and apoB levels (r=0.65, p<0.001), while no significant correlation was shown with Lp-a.

**3. Results**

**3.1. Patients' characteristics**

132 Carotid Artery Disease - From Bench to Bedside and Beyond

A total of 612 patients were evaluated, of which 123 fulfilled the eligibility criteria; finally, 100 had complete data to enter the analysis, 76 males and 24 females, median age 68 years (range 45-81). Diabetes mellitus was recorded in 26 of the 100 patients and hypertension in 66. Twenty patients had metabolic syndrome according to the national cholesterol education programmeadult treatment panel III (NCEP-ATP III) criteria [29]. Active smoking (defined as current of discontinued as far back as 5 years) was reported by 58 patients. Mean atorvastatin dose at baseline was 24.31±11.49 mg for group A and 20.62±10.39 mg for group B (p=0.1). By the end of the study period, the respective mean values were significantly increased to 30.45±16.27 mg

Each of the study group (A and B) comprised 50 patients. The two groups were comparable

Mean serum TC, LDL-cholesterol, TG, Lp-a, homocysteine, HDL-cholesterol, and oxLDL were significantly reduced at 12 months compared to baseline (table 2). Specifically, mean oxLDL dropped from 62.26±22.03 mU/l to 44.49±21.75 (p<0.001). A marked decrease was noticed

**Characteristic Total Group A Group B p value**

mean age in years ± SD 67.57±7.15 68.46±5.71 66.68±8.31 0.21 number of pts with DM (percentage) 37 ( 37% ) 18 (36%) 19 (38%) 0.83 number of pts with HTN (percentage) 67 ( 67% ) 36 (72%) 31 (62%) 0.29 number of smokers (percentage) 54 ( 54% ) 29 (54%) 25 (46%) 0.33 number of pts with CAD (percentage) 51 (51%) 24 (47%) 27 (53%) 0.55 mean ± SD total cholesterol (mg/dl) 232.23±47.8 235.24±49.2 229.22±46.7 0.53 mean ± SD LDL cholesterol (mg/dl) 151.27±41.7 154.16±42.8 148.84±40.9 0.52 mean ± SD HDL cholesterol (mg/dl) 51.97±12.7 52.12±12.1 51.82±13.4 0.9 mean ± SD triglycerides (mg/dl) 145.59±73.1 146.04±73.2 145.14±73.7 0.95 mean ± SD oxidized LDL (mU/l) 64.66±24.8 65.8±25.3 63.53±24.5 0.65 mean ± SD homocysteine (mU/l) 13.99±4.8 13.5±4.6 14.47±5.1 0.32

Pts: patients, DM: diabetes mellitus, HTN: arterial hypertension, MS: metabolic syndrome, SD: standard deviation, LDL:

males / females 72 / 28 36 / 14 36 / 14

low density lipoprotein, HDL: high density lipoprotein, CAD: coronary artery disease

**Table 1.** Study population baseline characteristics

for group A (p=0.044) and 28.75±17.57 mg for group B (p=0.007).

during the first 6 months and a plateau thereafter (Figure 5).

with regard of their baseline characteristics (table 1).

**3.2. Lipid profile and oxidised LDL**

Between smokers mean oxidized LDL was reduced from 60.68±24.09 mU/l at baseline to 45.84±24.89 mU/l at the end of study period (difference 14.84 mU/l, p = 0.0036). Similarly, between non-smokers it was reduced from 69.33±25.11 to 40.36±5.6 (difference 28.97, p<0.001). Non-smokers had approximately double decline of oxidized LDL levels compared to smokers. Carotid artery stenosis was reduced between smokers from 29.68±25.59% at baseline to 23.06±21.71% at 12 months (p = 0.002). Non-smokers also presented significant reduction of stenosis during the study period 24.67±26.22% vs 20±21.45%, p = 0.004). Non-smokers and smokers had similar decline of carotid stenosis in 12 months (6.61% vs 4.67%, Table 3).

TC: total cholesterol, LDL: low density lipoprotein cholesterol, oxLDL: oxidised LDL, TG: triglycerides, 0=baseline, 1=one month, 2=three months, 3=six months, 4=twelve months.

**Figure 5.** Time curve of change of total cholesterol, LDL cholesterol, triglycerides and oxidised LDL levels during the observation period.

In further analysis, the group of smokers was subdivided to mild (≤5 cigarettes/day), moderate (5 – 15 cigarettes/day) and heavy (≥15 cigarettes/day) smokers. The statistical significant reduction of oxidized LDL levels and degree of carotid stenosis was apparent in the subgroup of mild smokers (oxidized LDL at baseline 48.24±8.74 mU/l vs 41.54±9 mU/l at 12 months, p = 0.027 and stenosis at baseline 27.63±25.68% vs 23.42±21.74% at 12 months, p = 0.009), while it was not apparent in the subgroups of moderate and heavy smokers (oxidized LDL at baseline 86.82±37.7 mU/l vs 42.92±10.77 mU/l at 12 months, p = 0.077 and stenosis at baseline 34±31.9% vs 22±24.9% at 12 months, p = 0.186, for moderate smokers; respective values for oxidized LDL were 66.29±15.88 mU/l vs 34.81±5.48 mU/l, p = 0.06 and for stenosis 32.14±24.13% vs 22.86±22.8%, p = 0.174, for heavy smokers). The above described effect of smoking was taken into consideration during Cox-regression analysis.


0

tion coefficient r=0.7, p<0.001)

ox LDL (mg/dl) baseline 12 months difference

stenosis (%) baseline 12 months difference

Correlation of oxLDL change with stenosis change in 12 months

period, between smokers and non-smokers.

p<0.001

50 100 150 200 250 LDL

Correlation LDL with oxLDL

Oxidised Low Density Lipoprotein (LDL) Modification with Statin Therapy is Associated with...

http://dx.doi.org/10.5772/57188

135

Fitted values

**Figure 6.** Correlation of low density lipoprotein (LDL) with oxidised LDL (oxLDL) levels at baseline (Pearson's correla‐

LDL: low density lipoprotein cholesterol, oxLDL: oxidised LDL, CI: confidence intervals.

60.68±24.09 45.48±24.89 14.84

29.68±25.59 23.06±21.71 6.61

> Pearson's r = 0.412

ox\_LDL 95% CI

0.0036

0.002

0.021

**Table 3.** Comparison of mean oxidized LDL values and degree of carotid stenosis change during the 1 year follow-up

Within group B, the subgroup of patients with high degree of stenosis (>60%) had oxLDL 63.47±19.18 mU/l at baseline, while those with moderate and mild degree of stenosis (<60%) had 40.32±20.72 mU/l (p<0.001). Corresponding values at 12-months were 33.18±17.78 and

**Smokers P value Non Smokers P value**

69.33±25.11 40.36±5.6 28.97

24.67±26.22 20±21.45 4.67

> Pearson's r = 0.198

0.001

0.004

0.03

50

100

ox\_LDL/95% CI/Fitted values

150

LDL: low density lipoprotein, HDL: high density lipoprotein

**Table 2.** Comparison of mean ± standard deviation and respective p values of measured laboratory investigations at baseline and 12 months, in the total population, and the two groups.

LDL: low density lipoprotein cholesterol, oxLDL: oxidised LDL, CI: confidence intervals.

22.86±22.8%, p = 0.174, for heavy smokers). The above described effect of smoking was taken

232.23 ± 47.8 153.36±17.2 <0.0001

151.5±41.7 79.75±12.7 <0.0001

145.59 ± 73.1 111±53.1 0.0002

64.67±24.8 43.38±18.9 <0.0001

51.97±12.7 51.32±15.5 0.74

13.99±4.8 11.89±3.5 0.0006

156.57±26.7 160.35±25.3

129.95±31.3 77.1±11.8 <0.0001

25.08±23.8 27.72±29.1 0.48

**Table 2.** Comparison of mean ± standard deviation and respective p values of measured laboratory investigations at

0.3

235.24±49.1 154.24±16.9 <0.0001

154.16±42.8 79.54±13.2 <0.0001

146.04±73.2 112.1±54.7 0.01

65.8±25.3 42.16±17.6 <0.0001

52.12±12.1 52.22±16.3 0.97

13.5±4.6 11.88±3.8 0.057

156.46±27.3 162.02±23.7 0.28

131.84±31.4 77.58±13.1 <0.0001

25.67±24.1 29.42±29.8 0.49

229.22±46.7 152.48±17.7 <0.0001

148.84±40.9 79.96±12.3 <0.0001

145.14±73.7 109.9±51.96 0.0069

63.53±24.6 44.65±26.1 0.0007

51.82±13.4 50.42±14.8 0.62

14.48±5.1 11.9±3.4 0.0036

156.68±26.4 158.68±27.1

128.05±31.4 76.62±10.47 <0.0001

24.47±23.8 26.01±28.7 0.77

0.7

**Investigations Total Group A Group B**

into consideration during Cox-regression analysis.

134 Carotid Artery Disease - From Bench to Bedside and Beyond

total cholesterol (mg/dl) baseline total cholesterol (mg/dl) 12months

LDL cholesterol (mg/dl) baseline LDL cholesterol (mg/dl) 12months

triglycerides (mg/dl) baseline triglycerides (mg/dl) 12months

oxidized LDL (mU/l) baseline oxidized LDL (mU/l) 12months

HDL cholesterol (mg/dl) baseline HDL cholesterol (mg/dl) 12months

homocysteine (mg/dl) baseline homocysteine (mg/dl) 12months

apolipoprotein A (mg/dl) baseline apolipoprotein A (mg/dl) 12months

apolipoprotein B (mg/dl) baseline apolipoprotein B (mg/dl) 12months

lipoprotein a [Lp(a)] (mg/dl) baseline lipoprotein a [Lp(a)] (mg/dl) 12months

LDL: low density lipoprotein, HDL: high density lipoprotein

baseline and 12 months, in the total population, and the two groups.

p value

p value

p value

p value

p value

p value

p value

p value

p value

**Figure 6.** Correlation of low density lipoprotein (LDL) with oxidised LDL (oxLDL) levels at baseline (Pearson's correla‐ tion coefficient r=0.7, p<0.001)


**Table 3.** Comparison of mean oxidized LDL values and degree of carotid stenosis change during the 1 year follow-up period, between smokers and non-smokers.

Within group B, the subgroup of patients with high degree of stenosis (>60%) had oxLDL 63.47±19.18 mU/l at baseline, while those with moderate and mild degree of stenosis (<60%) had 40.32±20.72 mU/l (p<0.001). Corresponding values at 12-months were 33.18±17.78 and 38.81±29.02, representing a marked decline for patients with >60% initial stenosis and a far less decline for patients with <60% initial stenosis; yet the differences were not significant (table 4).

**3.6. The effect of LDL levels**

**4. Discussion**

was noticed past this time point.

should, ideally, comprise such measurements.

associated with additional improvement of stenosis.

Patients in group B who achieved LDL levels <70mg/dl during the observation period had a greater (28.08±28% vs 22.31±22.7%, difference 5.77%, p =0.06) reduction of carotid stenosis compared to those with LDL levels between 70 and 100 mg/dl (26.98±25.3% vs 21.35±21.3%, difference 5.63%, p < 0.001), but this difference was not statistically significant. Thus, in conservatively treated group B, further reduction of LDL than the limit of 100mg/dl was not

Oxidised Low Density Lipoprotein (LDL) Modification with Statin Therapy is Associated with...

http://dx.doi.org/10.5772/57188

137

This study demonstrates that atorvastatin administered in indivudualised doses, tittered to maintain serum LDL cholesterol levels <100mg/dl, significantly decreased lipid profile and oxLDL, reduced carotid artery stenosis in patients managed conservatively and prevented restenosis in patients with prior angioplasty. Oxidised LDL in this study correlated positively with the degree of carotid artery stenosis; it was also shown by multivariate analysis that oxLDL represented an independent risk factor for restenosis. To our knowledge this is the first prospective study with a long observation period of 12 months to report such a clear, significant reduction of oxLDL levels following atorvastatin therapy for carotid atheromatosis of various causes and to report an association of the degree of oxLDL reduction with remission of carotid stenosis. It is also of major importance that this robust, long-standing decline of oxLDL was achieved with doses of atorvastatin used in everyday clinical practice. Interestingly, this beneficial effect was completed in the first six months, while practically no further reduction

The mechanism by which statins modulate oxLDL levels has been controversial in the literature. Moreover, the association of oxLDL level modification with improvement of carotid atheromatosis and clinical outcome is not unequivocally established by large, double-blinded, randomised trials. Under this perspective, the present observational study provides reasona‐

Carotid intima media thickness (IMT) is a validated measure of carotid atherosclerosis. It is well established that carotid atherosclerosis, serves as an independent surrogate marker for CHD [30] and CVD [31]. Nevertheless, in the present study it was preferred to estimate the degree of carotid stenosis with a more direct approach, because this is more readily available in most hospital settings and because there is an obvious relation with clinical symptoms and signs. Besides, it represents a reliable method with sufficient reproducibility and it is practically the method of choice when evaluated patients candidate for endarterectomy or angioplasty. Evaluating carotid stenosis in turn, is an established method for estimating coronary risk [30] and cardiovascular risk [31]. Other parameters of vessel wall function, such as IMT and plaque morphology, even if clearly associated with cardiovascular risk in the literature, require well equipped laboratory and are not readily available in our hospital. Future research on the field

ble evidence that reducing oxLDL may independently improve carotid stenosis.


**Table 4.** Comparison of mean oxidized LDL levels at baseline and 12 months within patients of group Β (n = 50), according to degree of stenosis at enrollment.

#### **3.3. Anthropometrics**

Body mass index (BMI), weight, waist circumference and waist:hip ratio did not change significantly during the study period.

#### **3.4. Carotid stenosis**

Patients in group A had null stenosis at recruitment due to prior angioplasty with stenting. At the end of the 12-month statin therapy, no case of clinically important restenosis (>70%) was reported in this group (as restenosis was defined any increase of the carotid lumen diameter >5%). Patients in group B had mean percentage of stenosis at baseline 47.6±13.2%, which was significantly reduced following 12-month statin therapy (37.7±15.7%, p<0.001) (Table 5).


**Table 5.** Change of the percentage of carotid artery stenosis between baseline and 12months for patients in group B.

#### **3.5. Association of stenosis with oxidised LDL**

Group B patients in the highest quartile of oxLDL values had a 12-month risk ratio for restenosis of 1.025, 95%CI=1.006-1.044, p=0.0083 (figure 7). After adjusting for gender, age, smoking, SBP, TC, and LDL levels, these patients demonstrated a HR for restenosis of 4.319 compared with those in the lowest quartile (p<0.001, figure 7). This means that an increase of oxidized LDL by one unit increases the degree of carotid stenosis by 2.5%, for patients in group B. A weak but significant correlation was detected between oxLDL levels and the degree of carotid artery stenosis (r=0.17, p=0.018). Similar correlation was found between LDL choles‐ terol levels and carotid stenosis (r=0.18, p=0.0085). The strength of Pearson's correlation of mean oxidised LDL change with degree of carotid stenosis change during the 12-month period was greater for smokers compared to non-smokers (table 3).

#### **3.6. The effect of LDL levels**

38.81±29.02, representing a marked decline for patients with >60% initial stenosis and a far less decline for patients with <60% initial stenosis; yet the differences were not significant (table 4).

Mean oxidized LDL 63.47±19.18 mU/l 40.32±20.72 mU/l < 0.001

Body mass index (BMI), weight, waist circumference and waist:hip ratio did not change

Patients in group A had null stenosis at recruitment due to prior angioplasty with stenting. At the end of the 12-month statin therapy, no case of clinically important restenosis (>70%) was reported in this group (as restenosis was defined any increase of the carotid lumen diameter >5%). Patients in group B had mean percentage of stenosis at baseline 47.6±13.2%, which was significantly reduced following 12-month statin therapy (37.7±15.7%, p<0.001) (Table 5).

Mean % carotid stenosis ± standard deviation 47,6 ± 13,2 37,7 ± 15,7 0,001

**Table 5.** Change of the percentage of carotid artery stenosis between baseline and 12months for patients in group B.

Group B patients in the highest quartile of oxLDL values had a 12-month risk ratio for restenosis of 1.025, 95%CI=1.006-1.044, p=0.0083 (figure 7). After adjusting for gender, age, smoking, SBP, TC, and LDL levels, these patients demonstrated a HR for restenosis of 4.319 compared with those in the lowest quartile (p<0.001, figure 7). This means that an increase of oxidized LDL by one unit increases the degree of carotid stenosis by 2.5%, for patients in group B. A weak but significant correlation was detected between oxLDL levels and the degree of carotid artery stenosis (r=0.17, p=0.018). Similar correlation was found between LDL choles‐ terol levels and carotid stenosis (r=0.18, p=0.0085). The strength of Pearson's correlation of mean oxidised LDL change with degree of carotid stenosis change during the 12-month period

12 months Mean oxidized LDL 33.18±17.78 mU/l 38.81±29.02 mU/l NS

**Table 4.** Comparison of mean oxidized LDL levels at baseline and 12 months within patients of group Β (n = 50),

Baseline

**3.3. Anthropometrics**

**3.4. Carotid stenosis**

according to degree of stenosis at enrollment.

136 Carotid Artery Disease - From Bench to Bedside and Beyond

significantly during the study period.

**3.5. Association of stenosis with oxidised LDL**

was greater for smokers compared to non-smokers (table 3).

**Stenosis >60<70% Stenosis <60% P value**

**baseline 12 months p value**

Patients in group B who achieved LDL levels <70mg/dl during the observation period had a greater (28.08±28% vs 22.31±22.7%, difference 5.77%, p =0.06) reduction of carotid stenosis compared to those with LDL levels between 70 and 100 mg/dl (26.98±25.3% vs 21.35±21.3%, difference 5.63%, p < 0.001), but this difference was not statistically significant. Thus, in conservatively treated group B, further reduction of LDL than the limit of 100mg/dl was not associated with additional improvement of stenosis.
